Nucleic acid ligand and use thereof

Abstract

Described is a nucleic acid ligand, a mixture thereof, and the use thereof. The mixture contains two or more nucleic acid polymerase substrate analogs. The nucleic acid polymerase substrate analog is a single nucleic acid molecule or nucleic acid molecule analog which forms complementary pairing within a molecule, or a single or two nucleic acid molecules or nucleic acid molecule analogs which form complementary pairing between molecules; and a structure formed thereby has the characteristics of a nucleic acid polymerase substrate. The nucleic acid polymerase substrate analog is suitable for all polymerases and can be widely used in the field of nucleic acid amplification. The 3′ end of the nucleic acid ligand has a modification which inhibits the extension thereof.

1 . A nucleic acid ligand (nucleic acid polymerase substrate analog), wherein the nucleic acid ligand is a single nucleic acid molecule or nucleic acid molecule analog which forms intramolecular complementary pairing, or a single or two nucleic acid molecules or nucleic acid molecule analogs which form intermolecular complementary pairing; the nucleic acid ligand has a modification at 3′ end, which inhibits its extension; the nucleic acid ligand forms a stable structure with a nucleic acid polymerase when the temperature is maintained or below a certain temperature, and the enzyme activity of the nucleic acid polymerase is inhibited at this time, and when the temperature is higher than said certain temperature, the nucleic acid polymerase detaches from the nucleic acid ligand to exert its activity. 
     
     
         2 . The nucleic acid ligand according to  claim 1 , wherein the certain temperature is a temperature at which the nucleic acid polymerase exerts its activity. 
     
     
         3 . The nucleic acid ligand according to  claim 1 , wherein the number of the complementary pairing is 8-35. 
     
     
         4 . The nucleic acid ligand according to  claim 1 , wherein the modification is a dideoxy modification, a phosphorylation modification or an amino modification. 
     
     
         5 . The nucleic acid ligand according to  claim 1 , wherein the nucleic acid polymerase is a DNA polymerase or a RNA polymerase. 
     
     
         6 . The nucleic acid ligand according to  claim 5 , wherein the DNA polymerase is selected from the group consisting of the polymerases of Family A and Family B. 
     
     
         7 . The nucleic acid ligand according to  claim 5 , wherein the RNA polymerase is selected from the group consisting of reverse transcriptases of the AMV family or the MMLV family. 
     
     
         8 . (canceled) 
     
     
         9 . A method of nucleic acid amplification, comprising:
 step 1: contacting a sample to be tested containing a target nucleic acid with the following amplification reaction reagents to form a reaction mixture;   a) primers that can hybridize to the target nucleic acid;   b) a nucleic acid polymerase;   c) the nucleic acid ligand according to  claim 1 ;   d) a nucleoside triphosphate;   step 2: heating the reaction mixture to allow the paired nucleotides of the nucleic acid ligand to dissociate into a single strand and the nucleic acid polymerase to detach from the nucleic acid ligand and exert its activity, thereby forming a primer extension product.   
     
     
         10 . A nucleic acid amplification kit comprising the nucleic acid ligand according to  claim 1 . 
     
     
         11 . A nucleic acid extension reaction mixture comprising the nucleic acid ligand according to  claim 1 , a nucleic acid polymerase, at least one primer, a nucleic acid template, and a nucleoside triphosphate. 
     
     
         12 . A mixture of nucleic acid polymerase substrate analogs, wherein:
 a. containing two or more nucleic acid polymerase substrate analogs;   b. the nucleic acid polymerase substrate analog is a single oligomeric nucleic acid molecule or nucleic acid molecule analog which forms intramolecular complementary pairing, or a single or two oligomeric nucleic acid molecules or nucleic acid molecule analogs which form intermolecular complementary pairing; the nucleic acid polymerase substrate analog forms a structure which has the characteristics of a nucleic acid polymerase substrate;   c. the nucleic acid polymerase substrate analogs are modified at 3′ end, which inhibits their extension;   d. the two or more nucleic acid polymerase substrate analogs have different widths of temperature adaptation range;   e. when the temperature is maintained at or below a first temperature, the two or more nucleic acid polymerase substrate analogs are mixed with a nucleic acid polymerase and the two form a nucleic acid polymerase-substrate analog complex; at this time, the enzyme activity of the nucleic acid polymerase is significantly reduced relative to that in the absence of the nucleic acid polymerase substrate analog;   f. when the temperature is higher than the first temperature, the nucleic acid polymerase-substrate analog complex described in “e” disintegrates, and all or part of the nucleic acid polymerase activity is released.   
     
     
         13 . A mixture of a nucleic acid polymerase and a mixture of nucleic acid polymerase substrate analogs, wherein:
 a. containing two or more nucleic acid polymerase substrate analogs;   b. the nucleic acid polymerase substrate analog is a single oligomeric nucleic acid molecule or nucleic acid molecule analog which forms intramolecular complementary pairing, or a single or two oligomeric nucleic acid molecules or nucleic acid molecule analogs which form intermolecular complementary pairing; the nucleic acid polymerase substrate analog forms a structure which has the characteristics of a nucleic acid polymerase substrate, and can bind to a nucleic acid polymerase; the molecule number of each nucleic acid polymerase substrate analog is greater than the molecule number of the nucleic acid polymerase;   c. the nucleic acid polymerase substrate analogs are modified at 3′ end, which inhibits their extension;   d. the two or more nucleic acid polymerase substrate analogs have different widths of temperature adaptation range;   e. when the temperature is maintained at or below a first temperature, the two or more nucleic acid polymerase substrate analogs are mixed with a nucleic acid polymerase and the two form a nucleic acid polymerase-substrate analog complex; at this time, the enzyme activity of the nucleic acid polymerase is significantly reduced relative to that in the absence of the nucleic acid polymerase substrate analog;   f. when the temperature is higher than the first temperature, the nucleic acid polymerase-substrate analog complex described in “e” disintegrates, and all or part of the nucleic acid polymerase activity is released.   
     
     
         14 . The mixture according to  claim 12 , wherein:
 g. when the temperature is maintained at or below a second temperature, the nucleic acid polymerase substrate analog with a wide temperature adaptation range and the nucleic acid polymerase form a nucleic acid polymerase-substrate analog complex, and the nucleic acid polymerase substrate with a narrow temperature adaptation range cannot form a nucleic acid polymerase-substrate analog complex with the nucleic acid polymerase;   
       the first temperature is higher than the second temperature. 
     
     
         15 . The mixture according to  claim 14 , wherein there is a temperature difference between the first temperature and the second temperature, which is greater than or equal to 5° C. 
     
     
         16 . The mixture according to  claim 14 , wherein the width of the temperature adaptation range of the nucleic acid polymerase substrate analog is related to the number of its intramolecular or intermolecular complementary paired bases;
 preferably, when the temperature is maintained at or below the second temperature, the nucleic acid polymerase substrate analog having less complementary paired bases and the nucleic acid polymerase form a nucleic acid polymerase-substrate analog complex, but the nucleic acid polymerase substrate analog having more complementary paired bases cannot form a nucleic acid polymerase-substrate analog complex with the nucleic acid polymerase.   
     
     
         17 . The mixture according to  claim 16 , wherein, the number of complementary paired bases is from 8 to 35;
 preferably, the number of complementary paired bases is 10-30;   more preferably, the number of complementary paired bases is 10-20;   further preferably, the number of intramolecular complementary paired bases is 8-20, and the number of intermolecular complementary paired bases is 10-32.   
     
     
         18 . The mixture according to  claim 12 , wherein the 3′ end of the nucleic acid polymerase substrate analog is a non-OH group;
 preferably, modifications at the 3′ end of the nucleic acid polymerase substrate analog that inhibit its extension include dideoxy modifications, phosphorylation modifications, or amino modifications. 
 
     
     
         19 . The mixture according to  claim 13 , wherein the nucleic acid polymerase is a DNA polymerase or a RNA polymerase;
 preferably, the DNA polymerase is a thermostable DNA polymerase;   
       the RNA polymerase is a reverse transcriptase. 
     
     
         20 . (canceled) 
     
     
         21 . A method of nucleic acid amplification, comprising:
 step 1: contacting a sample to be tested containing a target nucleic acid with the following amplification reaction reagents to form a reaction mixture;   a) primers that can hybridize to the target nucleic acid;   b) a nucleic acid polymerase;   c) a mixture of nucleic acid polymerase substrate analogs according to  claim 12 ;   d) a nucleoside triphosphate, a deoxynucleoside triphosphate or a mixture thereof, or a nucleoside/deoxynucleoside triphosphate analog;   step 2: heating the reaction mixture to allow the paired nucleotides of the nucleic acid polymerase substrate analog to dissociate into a single strand and the nucleic acid polymerase to detach from the nucleic acid polymerase substrate analog and exert its activity, thereby forming a primer extension product.   
     
     
         22 . A nucleic acid amplification kit comprising the mixture according to  claim 12 . 
     
     
         23 . nucleic acid extension reaction mixture comprising the mixture according to  claim 12 , optionally a nucleic acid polymerase, at least one primer, a nucleic acid template; and a nucleoside triphosphate, a deoxynucleoside triphosphate or a mixture thereof, or a nucleoside/deoxynucleoside triphosphate analog.